Flumazenil decreases surface expression of α4β2δ GABAA receptors by increasing the rate of receptor internalization

Increases in expression of alpha 4 beta delta GABA(A) receptors (GABARs), triggered by fluctuations in the neurosteroid THP (3 alpha-OH-5 alpha[beta]-pregnan-20-one), are associated with changes in mood and cognition. We tested whether alpha 4 beta delta trafficking and surface expression would be altered by in vitro exposure to flumazenil, a benzodiazepine ligand which reduces alpha 4 beta delta expression in vivo. We first determined that flumazenil (100 nM-100 mu M, IC50 = similar to 1 mu M) acted as a negative modulator, reducing GABA (10 mu M)-gated current in the presence of 100 nM THP (to increase receptor efficacy), assessed with whole cell patch clamp recordings of recombinant alpha 4 beta 2 delta expressed in HEK-293 cells. Surface expression of recombinant alpha 4 beta 2 delta receptors was detected using a 3XFLAG reporter at the C-terminus of alpha 4 (alpha 4F) using confocal immunocytochemical techniques following 48 h exposure of cells to GABA (10 mu M)+THP (100 nM). Flumazenil (10 mu M) decreased surface expression of alpha 4F by similar to 60%, while increasing its intracellular accumulation, after 48 h. Reduced surface expression of alpha 4 beta 2 delta after flumazenil treatment was confirmed by decreases in the current responses to 100 nM of the GABA agonist gaboxadol. Flumazenil-induced decreases in surface expression of alpha 4 beta 2 delta were prevented by the dynamin blocker, dynasore, and by leupeptin, which blocks lysosomal enzymes, suggesting that flumazenil is acting to increase endocytosis and lysosomal degradation of the receptor. Flumazenil increased the rate of receptor removal from the cell surface by 2-fold, assessed using botulinum toxin B to block insertion of new receptors. These findings may suggest new therapeutic strategies for regulation of alpha 4 beta 2 delta expression using flumazenil. (C) 2015 Elsevier Inc. All rights reserved.